Prion diseases represent a diverse group of infectious neurodegenerative disorders. The most accepted hypothesis is that the infectious agent (termed prion) is a misfolded version of a normal protein completely devoid of nucleic acids. Disease is propagated when the infectious form (PrPsc) converts the normal form (PrPc) to the infectious form by reversibly combining with it. In scrapie, the prion is a glycoprotein with about a 30,000 MW protein core. To produce central nervous system (CNS) disease, PrPsc must enter the brain, which requires it negotiate the blood-brain barrier (BBB). The major goal of this research is to determine how PrPsc crosses the BBB and ultimately to develop therapeutic strategies for blocking passage into the CNS and so preventing prion disease. Work by us and others have shown that other neurotoxic glycoproteins (such as wheatgerm agglutinin and gp120, the coat of the AIDS virus) cross the BBB by inducing absorptive endocytosis (AE). We hypothesize that PrPsc crosses the BBBthrough the mechanism of AE. This hypothesis provides a mechanism for passage across the BBB of cell-free PrPsc and of PrPsc- infected immune cells and explains how some regions of the CNS, such as the thoracic spinal cord, can be especially targeted. Although our working hypothesis is that cell-free PrPsc is the major mechanism , these experiments are designed to determine the extent to which the other possible mechanisms of entry into the CNS (immune cell transfer, retrograde splenic nerve transmission, transmembrane diffusion, saturable carrier/receptor mediated transport, leakage via extracellular pathways) are operational for PrPsc. We will use highly purified, radioactively labeled PrPsc to determine rates of transport and distribution into brain regions, spinal cord, and CSF, the role of splenic nerves and immune cells in neuroinvasion, and in vitro models to examine the cellular biology of passage across the brain endothelial cell. Lay Summary: Prions cause rare, but devastating, diseases such as mad cow disease. To cause disease, prions must cross the blood-brain barrier to enter the brain. We will determine how prions cross the BBB. Knowing how prions enter the brain should lead to strategies on how to prevent prion diseases.